I. Host
A. Trichoderma reesei
The mesophilic filamentous fungus Trichoderma reesei is very efficient in secreting cellulase enzymes into the growth medium. In optimized cultivation conditions, amounts of up to 40 g/l of extracellular cellulase have been reported (Durand et al., Enzyme Microb. Technol. 10:341-346 (1988); Durand et al., in Biochemistry and Genetics of Cellulose Degradation, Academic Press, 1988, pp. 135-151).
Development of transformation systems for T. reesei (Knowles et al., EP244,234; Penttila et al., Gene 61:155-164 (1987); Berka et al., EP215,594) has made possible the application of genetic engineering methods to the fungus. By genetic engineering, production profiles of different cellulase enzymes have been modulated e.g., to give strains with improved levels of the endoglucanase I enzyme. The strong cbh1 promoter has been applied to promote endoglucanase expression (Nevalainen, H., et al., "The molecular biology of Trichoderma and its application to the expression of both homologous and heterologous genes," in Molecular Industrial Mycology, Leong and Berka, eds., Marcel Dekker Inc., New York, pp. 129-148 (1992); and Harkki, A. et al., Enzyme Microb. Technol. 13:227-233 (1991)).
In addition to tailoring the production profiles of homologous proteins, the production potential of T. reesei has been harnessed to express various heterologous proteins in the fungus. So far examples are few and include e.g., calf chymosin (Knowles et al., EP244,234; Berka et al., EP215,594; Harkki, A., et al., Bio/Technol. 7:596-603 (1989); Uusitalo, J. M., et al., J. Biotechnol. 17:35-50 (1991), CBH 1-Fab fusion antibodies raised against 2-phenyl-oxazolone (Nyyssonen et al., WO92/01797) and a fungal ligninolytic enzyme (Saloheimo, M. and Niku-Paavola, M. -L., Bio/Technol. 9:987-990 (1991)). For improved expression, the desired gene has been inserted into a cbh1 expression cassette and introduced into T. reesei by protoplast transformation (Harkki, A., et al., Bio/Technol. 7:596-603 (1989); Nyyssonen et al., WO92/01797; Saloheimo, M. and Niku-Paavola, M. -L, Bio/Technol. 9:987-990 (1991)). Even though heterologous filamentous fungal promoters such as Aspergillus amdS, argB and glucomylase (GA) can function in T. reesei at least to some extent (Penttila et al., Gene 61:155-164 (1987); Knowles et al., EP244,234) efficient expression requires the use of a homologous promoter. In addition, better yields have been obtained in some cases by producing the desired gene product as a fusion protein (Harkki, A., et al, Bio/Technol. 7:596-603 (1989); Nyyssonen et al., WO92/01791). The yields of heterologous proteins obtained from T. reesei have varied between 10-150 mg/l.
II. Glucoamylases
Glucoamylase enzymes (.alpha.-1,4,-glucan glucohydrolase, EC 3.2.1.3) are starch hydrolyzing exoacting carbohydrases. They are microbial enzymes and are produced extracellularly by many molds and some yeasts. Starch is a heterogeneous polysaccharide containing 15-30% amylose and 70-85% amylopectin. Amylose is a linear polymer of 500 or more .alpha.-D(1-4)-linked glucose residues. Amylopectin is a branched polymer composed of about 20-30 .alpha.-D(1-4)-linked glucose units, which are connected to each other through .alpha.-D(1-6) linkages. These branch points comprise 4-5% of the total glucosidic bonds in starch.
Glucoamylases hydrolyze both .alpha.-1,4 and .alpha.-1,6 linkages in polysaccharides such as starch, liberating glucose units from nonreducing ends of the polysaccharides. These two activities are distinct. By hydrolyzing .alpha.-1,4 and .alpha.1,6 glucosidic bonds, glucomylases liberate .beta.-D-glucose units from terminal nonreducing ends of a glucose polymer such as starch.
III. Debranching activities
Pullulanases are endo-acting hydrolytic enzymes specific for cleaving .alpha.-(1,6)-glucosidic bonds. However, the utility of pullulanases in many, industrial processes is limited by their inability to efficiently hydrolyze polymers smaller than maltosyl maltose. In contrast, some glucoamylases such as Hormoconis resinae glucoamylase P can hydrolyze a broad range of polymeric substrates including isomaltose (two glucose units connected by an .alpha.-(1,6)-glucosidic bond) and panose.
Glucoamylases differ considerably in their ability to hydrolyze .alpha.-(1,6)-glucosidic bonds. The highest debranching activities are found in the enzymes of fungi such as Hormoconis resinae (prior name Cladosporium resinae). Research on this fungus has revealed that it actually produces two distinct glucoamylases which exhibit different molecular masses and pI values. The smaller glucoamylase P, has a very high debranching activity whereas the larger glucoamylase S, has virtually no debranching activity as measured using pullulan (Fagerstrom et al., J. Gen. Microbiol. 136:913-920 (1990); McCleary & Anderson, Carbohydrate Research 86:77-96 (1980)).
IV. Isolation and Recombinant Expression of Glucoamylase
Several genes coding for various glucoamylases have been cloned and expressed either in yeast or fungal expression systems.
A procedure for purifying glucoamylase P as well as portions of its amino acid sequence have been published (Fagerstrom et al., J. Gen. Microbiol. 136:913-920 (1990)). In addition, a restriction map of two overlapping glucoamylase cDNA fragments has been published as well as evidence which suggests that recombinant laboratory yeast may be used to express this gene (Torkkeli et al., XIII International Specialized Symposium on Yeasts, Leuven, Belgium (1989)).
The glucoamylase genes from both Aspergillus niger (WO 86/07091; WO 88/09795; U.S. Pat. No. 5,024,941) and from Aspergillus awamori (U.S. Pat. No. 4,794,175 and EP Patent No. 126206) have been cloned and expressed in yeast cells.
The glucoamylase gene from a fungus of the genus Rhizopus has been expressed in Saccharomyces cerevisiae (EP Application Publication No. 186066). In addition, other types of amylolytic yeast have been constructed by the introduction of a modified Rhizopus glucoamylase gene (Ashikari et al., App. Microbiol. and Biotech. 32:129-133 (1989)).
A recombinant Saccharomyces has been constructed by transforming S. cerevisiae with a glucoamylase gene from C. albicans resulting in the secretion of the .alpha.-(1,4)-glucosidic bond-cleaving enzyme (EP Patent Application Publication No. 0362179).
The glucoamylase gene from Schwanniomyces castellii has been cloned and expressed in Saccharomyces cerevisiae as well as in other forms of yeast (EP Patent Application Publications No. 0260404 and No. 0257115).
Brewing yeasts have been transformed with a recombinant plasmid having a gene coding for a glucoamylase of Saccharomyces diastaticus (Park et al., MBAA Technical Quarterly 27:112-116 (1990)).
An amylolytic S. cerevisiae strain which is able to use starch as its sole source of carbon has been developed and used to express the .alpha.-amylase and glucoamylase genes of Schwanniomyces occidentalis (Hollenberg and Strasser, Food Biotechnology 4:527-534 (1990)).
Dohmen et al., Gene 95:111-121 (1990), have expressed a Schwanniomyces occidentalis glucoamylase in S. cerevisiae cells by transforming the cells with centromere plasmids carrying the glucoamylase gene fused to different S. cerevisiae promoters.
The amino acid sequences of several different microbial glucoamylases have been determined. The complete sequences of Aspergillus niger (Svensson et al., Carlsberg Res. Comm. 48:529-544 (1983)) and Aspergillus awamori (Nunberg et al., Mol. and Cell. Biol. 4:2306-2315 (1984))glucoamylases are identical. The glucoamylase enzymes from Rhizopus oryzae (Ashikari et al., Agricultural and Biological Chem. 50:957-964 (1986)), Saccharomyces diastaticus (Yamashita et al., J. Bacteriol. 161:567-573 (1985)), Saccharomyces cerevisiae (Yamashita et al., J. Bacteriol. 169:2142-2149 (1987)); Aspergillus shirousami (Shibuya et al., Agric. Bio. Chem. 54:1905-1914 (1990)); Schwanniomyces occidentalis (Dohmen et al., Gene 95:111-121 (1987)); Clostridium sp. G0005 (Ohnishi et al., Eur. J. Biochem. 207:413-418 (1992)); and Saccharomycopsis fibuligera (Itoh et al., J. Bacteriol. 169:4171-4176 (1987)) have also been sequenced.
V. Industrial Uses of Glucoamylases
The most widely used organism for commercial alcohol fermentation, Saccharomyces cerevisiae, cannot directly use starch as a growth substrate because S. cerevisiae lacks the enzymes necessary to hydrolyze the starch polymer to fermentable monomeric units. As a result, starch must undergo a pre-fermentation hydrolysis process before it can be used for large-scale ethanol production.
Typically, in the ethanol process, the starch polymer is ground, gelatinized by heating and then liquified by .alpha.-amylase, an endo-acting enzyme which hydrolyzes .alpha.-(1,4)-glucosidic bonds. As a result, .alpha.-limit dextrins containing .alpha.-(1,6)-glucosidic bonds are formed. Currently, cleavage of these .alpha.-(1,6)-glucosidic bond linkages constitutes the main rate limiting step in the complete hydrolysis process and industrial use of substrates containing these bonds.
Efforts have been made to enhance the efficiency of this process through improved enzymatic protocols for the treatment of starch. For example, the glucoamylase most commonly used in ethanol production comes from Aspergillus. However, the debranching activity of this enzyme is often not sufficient for production purposes and preparations must be supplemented with other enzymes such as pullulanases. Pullulanases are ineffective in degrading small .alpha.-limit dextrins.
U.S. Pat. No. 4,211,842 and U.S. Pat. No. 4,234,686 present a method for obtaining a mixture of starch degrading enzymes from Cladosporium resinae (former name for Hormoconis resinae), and a procedure for isolating a glucoamylase with high debranching activity from the mixture. U.S. Pat. No. 4,318,927 suggests producing a low caloric alcoholic beverage using a mixture of starch-degrading enzymes recovered from the culture medium of Cladosporium resinae (ATCC No. 20495). Others have described beer production, especially low-calorie (light) beer wherein the unfermentable carbohydrate dextrin is broken down into fermentable sugars by glucoamylase enzymes of different origin (U.S. Pat. No. 4,684,525, World Food & Drink Report (WFD), Jan. 5, 1987), Chemical Marketing Reporter, Apr. 7, 1986, p.121). U.S. Pat. No. 4,863,864 and EP 185,327 describe alcohol production from starch using glucoamylase. U.S. Pat. No. 4,898,738 describes sake production using Aspergillus glucoamylase.
In addition to manufacturing processes directed to the production of alcohols or alcoholic beverages, glucoamylases are useful in a wide variety of applications requiring the hydrolysis of raw starch, or the presence of a debranching activity. For example, such applications include starch analysis (Rickard, J. E. et al., J. Sc. Food and Agricul. 41:373-379 (1987); the manufacture of glucose syrups (Illanes, A., Alimentos 8:22-29 (1983)), high-DE glucose syrups and high-maltose syrups (EP 405,283, Shen, G. J. et al., Appl. Microbiol. Biotech. 33:340-344 (1990)), the production of isomaltose (U.S. Pat. No. 4,898,820); the hydrolysis of maltose and maltodextrins (Celebi, S. S. et al., J. Appl. Biochem. Biotechnol. 27:164-171 (1991)); the preparation of high purity dextrose (EP452,238, JP 3,139,289); high maltotetraose and high maltose content starch hydrolysates (U.S. Pat. No. 4,971,906, U.S. Pat. No. 4,925,795); straight linear dextrin for use in food, medicines and cosmetics (JP 2049594); rice preparations that lack stickiness (JP 2031652); the preparation of food fibers by the enzymatic treatment of seed husks or brans, such as, for example, corn hull hydrolysis or maize husks, rice husks, soy bean husks, skins of peanuts, and brans of rice, wheat, barley, oat, adlay, rye, (JP 2101016), and especially the elimination of serum cholesterol elevation factor from wheat bran (JP 63185931); highly stable emulsions useful in the preparation of chocolate (EP 135,768B); the assay of starch contents of various biological materials, the assay of .alpha.-amylase (U.S. Pat. No. 4,902,621); the production of polysaccharides with improved rheological properties over raw starch (AU 8826548); the production of crystalline 2-O-alpha-D-glycopyranosyl-L-ascorbic acid (EP 425,066); lubricants and gels (EP 372,184); the synthesis of branched cyclodextrin (U.S. Pat. No. 4,931,389, Yoshida, Y. et al., Hakko Kogaku Kaishi 68:197-203 (1990)); an additive to laundry and dish washing detergents (U.S. Pat. No. 5,020,377, EP 418,835, EP 450,627, EP 425,397); use in enzyme electrodes and multi-enzyme electrodes (Hamid, J. A. et al., Analyst 115:1289-1295 (1990)); use of fragments containing the starchbinding domain for the preparation of genetically engineered peptide affinity tails for the recovery of fusion proteins (Ford, C. et al., J. Cell. Biochem. (Suppl.) 14D:30 (1990), Chen, L. et al., Abst. Annu. Meet. Am. Soc. Microbiol. 90:269 (1990)); wood and textile industry applications such as, for example, the preparation of plywood adhesives and particle board binders (Mukherjee, S., Brazil Patent No. 400/88, issued Sep. 5, 1989) and saccharification of lignocellulosic materials; the preparation of high solids dextrin adhesives for the high speed coating of paper and wrinkle-free conversion of paper to envelopes, poster board, etc. (U.S. Pat. No. 4,921,795); the preservation of protein-containing animal or vegetable fodder (EP 346,909); the production of polysaccharides such as xanthine by aerobic fermentation of microorganisms wherein fermentation occurs in the presence of starch and an amylolytic enzyme such as glucoamylase (EP 319,372); improving the filterability of glucose syrups and/or lowering the viscosity of the same (U.S. Pat. No. 4,746,517); lowering the viscosity of organic slurries in fermentation processes (Wu, Y. V. et al., J. Agric. Food Chem. 37:1174-1177 (1989); the bioconversion of distillery wastes (Perdih, A. et al., Enzyme Microb. Technol. 13:848-852 (1991) or vegetable wastes (Von Richter, G. Starch 35:113-118 (1983)) or fruit waste (Horn, C. H. et al., Biol. Wastes 24:127-136 (1988) into feedstuff including feed additives, bulk fillers, sweetening agents, liquid feed components for farm animals, raw materials for ethanol production for consumption and as a fuel source); the preparation of feedstuff from meat by-products such as slaughterhouse, leather and good processing industry wastes (NL 8403620); treatment of feeds and fodder to increase contents of reducing sugars (SU 869745), improved utilization of sugar present in such feedstuff (Panciroli, G., Suinicoltura 24:19-24 (1983) including grass hays, mixed hays, legume hays, corn silage and rice straw (Abe, A. et al., J. Anim. Sci. 48:1483-1490 (1979)); and assays to predict the digestibility of animal feeds (Dowman, M. G. et al., J. Sci. Food Agric 33:689-696 (1982)). When used as a feed additive for food animals, the recombinant bacterial biomass may be added directly to the animal's fodder (SU 916,336).
Research has also been directed to the discovery, cloning and expression of enzymes with greater hydrolysis efficiency. A method of preparing glucoamylase S by culturing Cladosporium resinae (ATCC No. 20495) has been presented in U.S. Pat. No. 4,318,989.
Fagerstrom et al., J. Gen. Microbiol. 136:913-920 (1991) have presented evidence which suggests that of all the currently available glycoamylase enzymes, H. resinae glucoamylase P has the most favorable characteristics for industrial use because it is characterized by having an exceptionally high debranching activity. Thus, glucoamylase P may eliminate the need for the supplemental debranching enzymes in many industrial processes. However, H. resinae produces glucoamylase P in only minimal amounts (U.S. Pat. No. 4,318,927) and therefore, cannot be used efficiently in ethanol production. Therefore, a need still exists for an economical, large scale production of glucoamylase with a high debranching activity.